The aim of this application is to develop novel contrast agents functionally specific to Cerenkov imaging. Cerenkov light emission occurs when beta particles emitted during radioactive decay exceed the speed of light in a dielectric medium. Cerenkov emission is multispectral and continuous across the visible wavelengths. A substantial fraction of the photons released are at wavelengths greater than 600 nm and as a result Cerenkov radiation can be detected using optical imaging techniques routinely used in small animals. This could be used to add functional capabilities to positron emission tomography (PET) imaging but can also act as a stand-alone imaging modality. We propose here Cerenkov specific contrast agents that report on function based on selective bandwidth quenching. The absorption of a selected band of photons by a functional chromophore attenuates the emission within a defined wavelength range and allows the measurement of a specific activity using a radiolabeled probe. To demonstrate this we synthesized prototype 18F-labeled pH-responsive Cerenkov probes based on standard pH indicators. A transition from acidic to basic causes a color change that selectively absorbs a band of photons, reducing detected Cerenkov emission. This selective bandwidth quenching can be achieved intermolecularly or intramolecularly and can be measured in vitro and in vivo. Moreover, ratiometric imaging at different wavelengths can report not only on the function imparted by the dye but also estimate other parameters including the depth of the probe. In this application we will synthesize and test Cerenkov specific contrast agents for detection of pH and redox status in tumors. Although the concept of selective bandwidth quenching is generally applicable, we chose these because they are important measures in tumor biology and because there are known dyes and studies available to validate our methods. In Aim 1 we will synthetically optimize the pKa, ?max and lipophilicity of the pH sensitive dyes for in vivo detection of tumor pH and test these compounds in a sophisticated murine breast cancer model where lactate dehydrogenase has been silenced. In Aim 2, we will increase specificity by optimizing acquisition parameters for ratiometric imaging and sensitivity by designing DOTA-chelated 90Y probes to increase detected signal by 20-30 fold. In Aim 3, we will extend the overall concept by designing Cerenkov specific contrast agents for the detection of redox status in vitro and in vivo. The modulation of Cerenkov emission using selective bandwidth quenching defines a new imaging platform that can be used for the direct imaging of non-fluorescent chromophores in vivo. There are numerous dyes that functionally alter their absorption in response to chemical or physiological stimuli and many of these absorb at wavelengths appropriate for Cerenkov imaging. The successful development of this platform will allow the creation a wide range for contrast agents for dual Cerenkov-PET imaging to comprehensively describe tumor environment, metabolism and treatment.